506153.v2
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PHA productivity and yield of Ralstonia eutropha when intermittently or continuously fed a 2
mixture of short chain fatty acids. 3
4
Panchali Chakraborty 5
1199 Edison Drive 6
Cincinnati, OH-45216 7
Phone: 859-402-5258 (C), 513-948-5015 (O) 8
Email: [email protected] 9
William R. Gibbons 10
Department of Biology and Microbiology 11
South Dakota State University, Brookings, SD-57006 12
Phone: 605-688-5499, Fax: 605-688-6675 13
E-mail: [email protected] 14
Kasiviswanathan Muthukumarappan1 15
Department of Agriculture and Biosystems Engineering 16
SAE 225/ Box 2120, South Dakota State University, Brookings, SD-57006 17
Phone: 605-688-5661, Fax: 605-688-6764 18
E-mail: [email protected] 19
20
1 Corresponding author: Department of Agricu lture and Biosystems Engineering
SAE 225/Box2120, South Dakota State University, Brookings, SD-57006
Phone : 605-688-5661, Fax: 605-688-6764
2
ABSTRACT 21
The research described in this present study was part of a larger effort focused on 22
developing a dual substrate, dual fermentation process to produce Polyhydroxyalkanoate 23
(PHA). In the first bioreactor Ralstonia eutropha (R. eutropha) was rapidly grown to a high 24
cell density on a low cost medium. The substrate evaluated in this study for the growth phase 25
of R. eutropha was condensed corn solubles (CCS), a low-value byproduct of the dry-mill, 26
corn ethanol industry. In the second bioreactor a mixed culture of rumen microbes would 27
metabolize another feedstock (lignocellulosic biomass or other low cost byproduct) into a 28
mixture of Short Chain Fatty acids (SCFAs), called the Artificial Rumen Fluid (ARF). This 29
ARF would subsequently be fed to the R. eutropha cell mass to drive PHA production. The 30
focus of this study was developing and optimizing a strategy for feeding a mixture of SCFAs 31
(simulated ARF) and maximizing PHA production in a cost-effective way. SCFAThree 32
different feeding strategies were examined in this study. The culture was grown to high cell 33
densities in nitrogen supplemented CCS media in 5 L bioreactors. After 48 h of incubation at 34
30ºC, ARF was added to the bioreactors using the following schemes : 1) 124 ml at 48, 72, 35
and 96 h, 2) 20 ml every 3 h from 48-109 h. and 3) 7.75 ml h-1 from 48-96 h continuously. 36
ARF was not inhibitory to the growth of the organism since the organism continued to grow 37
after its addition. The overall growth rate of R. eutropha was 0.2 h-1. The second ARF feeding 38
strategy gave the best results in terms of PHA production. PHA productivity (0.0697 g L-1 h-39
1), PHA concentration (8.37 g L-1) and PHA content (39.52%) were the highest when ARF 40
was fed every 3h for 61h. This study proved that CCS, can be potentially used as a growth 41
medium to boost PHA production by R. eutropha thus reducing the overall cost of biopolymer 42
production. 43
3
Key words: Artificial Rumen Fluid, Bioreactor, Condensed Corn Solubles, 44
Polyhydroxyalkanoate, Ralstonia eutropha, Short chain fatty acids. 45
INTRODUCTION 46
Biodegradable polymers made from renewable resources such as agricultural wastes, corn, 47
cassava, tapioca, whey, etc., do not lead to depletion of finite resources. The most studied of 48
the biodegradable polymers include polyesters, polylactides, aliphatic polyesters, 49
polysaccharides, and various copolymers [3]. These biopolymers have many of the desirable 50
physical and chemical properties of conventional synthetic polymers [1]. To this point, high 51
production costs have limited the use of biopolymers. However if these costs can be reduced, 52
there would be widespread economic interest [21]. 53
The focus of this project was production of the biopolymer 54
Polyhydroxyalkanoate (PHA) at a low cost. PHA is actually a term used to describe a diverse 55
family of polymers that are composed of 3-hydroxy fatty acid monomers. The carboxyl group 56
of one monomer forms an ester bond with hydroxyl group of the neighboring monomer. 57
Polyhydroxybutyrate (PHB) has been studied in most detail. PHB has good oxygen 58
impermeability, moisture resistance, water insolubility, and optical purity [10, 17]. Young's 59
modulus and tensile strength of PHB are similar to polypropylene, but elongation at break is 6 60
% as opposed to that of 400 % for polypropylene [22]. It has good UV resistance, but poor 61
resistance to acids and bases [9]. The oxygen permeability is very low, making PHB a suitable 62
material for use in packaging oxygen-sensitive products. PHB has low water vapor 63
permeability compared to other bio-based polymers but higher than most standard polyolefins 64
and synthetic polyesters [8, 17]. Since PHB is toxicologically safe, it can be used for articles 65
which come into contact with skin, feed or food [6]. 66
4
In the food industry, PHA has a wide application as edible packaging material, coating 67
agent, flavor delivery agent and as dairy cream substitute [34, 35]. It can also be used for 68
making bottles, cosmetics, containers, pens, golf tees, films, adhesives and non-woven 69
fabrics, toner and developer compositions, ion- conducting polymers and as latex for paper 70
coating applications [24, 30]. It can be used to make laminates with other polymers such as 71
polyvinyl alcohol. The degradation products of PHB are found in large concentrations in 72
human blood plasma, so is not toxic for human use [15]. 73
PHA production by Ralstonia eutropha (R. eutropha) generally occurs during 74
stationary phase. Hence cells are first grown to high density, after which a key nutrient is 75
limited to trigger PHA synthesis [15]. Because of this dual phase process, PHA production 76
lends itself to fed-batch, as well as, continuous operation. This follows Pontryagin’s 77
maximum principle, which is an optimal feeding strategy for fed-batch fermentation [29]. The 78
key to this principle is determining the optimum switching time (tc). The maximum growth 79
rate (μmax) should be initially maintained, then switched to the critical growth rate (μc) at tc to 80
maximize the specific product production rate (ρmax). Since growth rate is affected by Carbon 81
: Nitrogen (C:N) ratio, it should also be changed at tc. 82
A variety of carbon sources have been used for production of PHA using different 83
fermentation strategies. Carbohydrates, oils, alcohols, fatty acids, and hydrocarbons are 84
potential carbon sources for PHA production. Ethanol byproducts, cane and beet molasses, 85
cheese whey, plant oils, hyrolysates of corn, cellulose, hemicellulose, palm oil, soybean oil, 86
tallow, corn steep liquor, casamino acids, and food scraps had been used as substrates to 87
produce PHA using different organisms [7, 12, 13, 18, 20, and 31]. 88
5
The substrate evaluated in this study for the growth phase of R. eutropha was 89
Condensed Corn Solubles (CCS), which is a low-value byproduct of the dry-mill, corn 90
ethanol industry. In the dry mill process, the whole corn is milled, mixed with water, and 91
enzymatically hydrolyzed to convert starch to glucose, which is converted to ethanol by 92
fermentation. After distillation to remove ethanol, the larger corn particles are recovered by 93
centrifugation as distiller's wet grains. The supernatant is condensed in multiple-effect 94
evaporators to give condensed corn solubles [11]. 95
The composition of CCS is shown in Table1. Corn-milling byproducts are typically 96
marketed as animal feed because of their protein content. However these byproducts may also 97
contain residual carbohydrates, which might be utilized by microbial fermentation to produce 98
industrial biopolymers. CCS is an excellent source of vitamins and minerals, including 99
phosphorous and potassium. 100
The objective of this study was to determine the effects of adding Artificial Rumen Fluid 101
(ARF) on cell viability, growth as well as PHA production to a 48 h culture of R. eutropha. 102
Different strategies of feeding simulated ARF into the bioreactors were assessed to maximize 103
PHA production. 104
MATERIALS AND METHODS 105
Culture, Maintenance and Inoculum Propagation 106
The ATCC 17699 type strain of R. eutropha was used. The culture was routinely 107
transferred to nutrient broth and incubated on a reciprocating shaker (250 rpm) at 30ºC for 24 108
h. For short term maintenance the culture was stored on Tryptic Soy Agar (TSA) slants 109
covered with mineral oil and stored in the refrigerator. Inoculum for all trials was prepared in 110
a stepwise manner, by transferring the culture from TSA plates into 100 ml of the CCS 111
6
medium (described below), then incubating for 24 h on a rotary shaker (250 rpm) at 30°C. 112
The inoculum rate for all bioreactor trials was 1 % (v v-1) from a 24 h grown culture to an 113
average OD of 1.04. 114
Medium 115
A low-cost medium based on CCS was developed in a prior study [4]. This medium, 116
containing 240 g CCS L-1, with a C:N ratio of 50:1 was the best medium for the growth of R. 117
eutropha. The medium was prepared by mixing 1,370 ml CCS with 4,630 ml deionized water, 118
adjusting the pH to 6.5 using 10 M sodium hydroxide (NaOH), then centrifuging at 11,000 119
rpm for 7 min at 15-25ºC. The supernatant was then filtered through Whatman filter paper 120
#113 and autoclaved. A filter sterilized 178 g L-1 ammonium bicarbonate (NH4HCO3) stock 121
solution was prepared and then 20.4 ml of this solution was added to each liter of CCS 122
medium to adjust the C:N ratio to 50:1. The pH was further adjusted to 7.0 by adding 10 N 123
sulfuric acid (H2SO4) before inoculation. 124
Fermentation Conditions for Cultivation of R eutropha in Bioreactor 125
Experimental trials were conducted in 5 L New Brunswick, Bioflo III, Edison, NJ 126
bioreactor that contained 4 L of CCS medium. Filter sterilized air (1 L L -1 min-1) was sparged 127
into the bioreactor, and 2-3 ml of antifoam (Cognis Clerol FBA 5059, Cognis, Cincinnati, 128
OH) were added to the medium before inoculation. Fermentation medium was incubated at 129
30°C and 500 rpm for 48 h, since prior research had shown R. eutropha to reach its maximum 130
population by this time/temperature combination [4]. 131
At 48 h we began feeding the fermenter a mixed Short Chain fatty Acid (SCFA) 132
solution, using any of the three different strategies. This SCFA solution, referred to as ARF, 133
contained 10 parts acetic, 2 parts butyric, 15 parts lactic, and 20 parts propionic acids (v v-1). 134
7
The composition of ARF was based on a separate study evaluating fermentation of biomass 135
with rumen consortia. A total volume of 372 ml of ARF was fed to the culture over a period 136
of 48 h. In the first feeding strategy (24h feeding), 124 ml of ARF was added at 48, 72, and 96 137
h. In the second feeding strategy (3h feeding), 20 ml ARF was added at 48 h and then every 3 138
h until 109 h. In the third feeding strategy (continuous feeding), ARF was continuously added 139
from 48 to 96 h at a rate of 7.75 ml h-1. All incubations were continued until 144 h. Two 140
replications were performed for each feeding strategy to determine the effect of mixed fatty 141
acids on cell viability, acid utilization, and PHA production. 142
Analytical Methods 143
Viable Counts, Cell Dry Weights, pH, HPLC and Ammonium/Phosphate Analysis 144
Samples were collected every 12h and viable cell counts were done with TSA. At 72, 145
96, and 144 h, 50 ml samples were collected to determine cell dry weights. Samples were 146
centrifuged and the precipitate was dried in the hot air oven at 80°C for 2 days. pH was 147
measured using an Acumet 950 pH meter (Thermo Fisher Scientific of Waltham, MA). 148
Samples were also analyzed via a Waters HPLC system (Milford, MA) for sugars, organic 149
acids and glycerol. These samples were first filtered through a non-sterile 0.2 μm filter to 150
remove solids, and then frozen until analysis. An Aminex HPX 87H column (Bio-Rad 151
Laboratories, Hercules, CA), operated at 65ºC with a helium-degassed, 4 mM H2SO4 mobile 152
phase at a flow rate of 0.6 ml min-1 was used. Peaks were detected using a refractive index 153
detector. Standard solutions of maltose, glucose, lactic acid, butyric acid, acetic acid, 154
propionic acid, succinic acid, and glycerol (at 3 and 30 g L-1) were used to calibrate the 155
integrator. Samples collected at 0, 72, and 120 h were also tested for ammonia and phosphate 156
using Hach Ammonium and Phosphate Unicell tests (Hach Company, Loveland, Colorado). 157
8
PHA Analysis 158
To measure PHA, 50 ml samples of broth were collected at 72, 96, and 144 h and 159
centrifuged. The pellets were then lyophilized and ground using a mortar and pestle. The 160
method developed by Braunegg et al. [2] was used to simultaneously extract and derivatize 161
PHA to the 3-hydroxyalkanoate methyl esters of the monomers. In this method, 20-30 mg of 162
ground cells were digested by adding 5 ml of digest solution and incubating at 90-100ºC for 4 163
h. The digest solution contained 50% chloroform, 42.5% methanol, 7.5% H2SO4 (v v-1). 164
After cooling, sample was washed with 2 ml of water, and the bottom layer (containing the 165
chloroform and methyl esters of PHA) was collected and placed in a Gas Chromatography 166
(GC) vial with anhydrous sodium sulfate (to remove residual water). Vials were frozen until 167
analysis. 168
PHA was quantified using a Hewlett-Packard 5890 Series II (Palo Alto, CA) GC with 169
a flame ionization detector (GC-FID) [2, 5]. Split injection was used onto a Supelco SSP-170
2380 (Park Bellefonte, PA) capillary column (30 m x 0.25 mm I.D. with 0.20 um film). The 171
inlet head pressure was maintained at 28 psi and the temperature program started at 50°C for 172
4 min, then increased by 3ºC min-1 to a final temperature of 146°C for 4 min. The injector 173
and detector temperatures were 230ºC and 240ºC, respectively. Purified poly (3-174
hydroxybutyric acid co-3-hydroxyvaleric acid) (P(HB-HV)) obtained from Sigma-Aldrich 175
was used for a standard calibration. The co-polymer consisted of 88 % 3-hydroxybutyric acid 176
(3-HB) and 12 % 3-hydroxyvaleric acid (3-HV). Co-polymer concentrations of 2-10 mg ml-1 177
were digested as above, and then analyzed by GC-FID. Retention times were 14.9 min for 178
methylated 3-HB, and 17.8 min for methylated 3-HV. 179
180
9
Statistical Analysis 181
All trials were performed in duplicates. Various fermentation parameters (maximum 182
cell populations, SCFA utilization rates, PHA productivity, etc) were analyzed to determine 183
least significant differences between treatments using randomized complete block design. 184
Fermentation Efficiency (FE) was calculated as the percentage of substrate supplied to that 185
was consumed. PHA productivity was calculated as the concentration of PHA produced per 186
hour whereas PHA content was determined as a percentage of PHA concentration over cell 187
dry weight. Data were analyzed using the PROC GLM procedure of SAS software to 188
determine F values and Least squares (LS) means. Exponential regression equations were 189
used to determine growth rates and acid utilization rates for each replication, from which 190
means were calculated. They were statistically analyzed by ANCOVA to test homogeneity of 191
slopes. Statistical data were analyzed at the significant level of p<0.05. 192
RESULTS 193
In all bioreactor trials the organism was incubated under similar conditions (30 º C, 194
500 rpm and aeration at 1L L-1 min-1) for the first 48 h, and this data was relatively uniform. 195
Figure 1 and Table 2 show the average cell population, growth rate, acid utilization, and 196
ammonia and phosphate uptake rates during this growth period. 197
Compared to shake flasks trials (Table 3), the maximum cell population in bioreactor 198
trials was almost 10 fold higher (2.3 X 1010 cfu ml-1). Likewise, the growth rate of R. 199
eutropha was also higher in the bioreactor (0.20 h-1) compared to the aerated shake flasks 200
(0.13 h-1). In the shake flask trials, lactic acid was consumed at the fastest rate followed by 201
acetic, butyric, succinic, and propionic acids. In the bioreactors, the organic acid utilization 202
rates in the growth phase were generally similar to shake flask trials except for lactic acid. It 203
10
was consumed slower than acetic acid. Overall acid utilization rates were higher in the 204
bioreactors than in shake flasks probably due to the higher cell population. Percentage acid 205
consumptions were also higher in the bioreactor with the exception of lactic acid. Ammonia 206
and phosphate usage rates were higher in bioreactor trials again due to the higher cell 207
population. 208
Three ARF feeding strategies were compared in this study. Figures 2-4 shows that 209
ARF feeding resulted in a slight increase in cell populations. The effects of different feeding 210
strategies on maximum cell population, and ammonia and phosphate utilization rates are 211
shown in Table 4. The maximum cell populations were not significantly different between the 212
three treatments. In all cases the cell population continued to rise after ARF additions began, 213
suggesting that the acid levels were not inhibitory. The rates of ammonia utilization and 214
phosphate utilization were similar for all feeding strategies. 215
As expected, the 24 h addition method (Figure 2) resulted in the highest spikes in 216
SCFA concentration, with acid levels then falling as R. eutropha metabolized the acids to 217
PHA. There was no apparent accumulation of lactic, butyric, or succinic acids for the 24h 218
feeding strategy. However, acetic acid (2.3 g L-1), and to a lesser extent propionic acid (0.5g 219
L-1), had accumulated over time. All the SCFAs were completely utilized by 144 h for the 3h 220
feeding strategy. For the continuous feeding strategy all the SCFAs were consumed by 144h 221
with the exception of propionic acid (0.5g L-1). 222
Utilization rates of the individual SCFAs during the final 96 h of fermentation (48 223
to144 h), along with the combined acid utilization rates, are shown in Table 4. The combined 224
utilization rate also included consumption of succinic acid that was already present in the 225
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CCS medium. The highest combined acid utilization rates were observed in the 3 h and 226
continuous feeding strategies, with the lowest rate in the 24 h feeding method. 227
The trend of higher acid utilization rates with the 3 h and continuous feeding methods 228
was also evident for the individual acids. However the difference was only significant for 229
acetic acid. Propionic and lactic acids were used most rapidly followed by acetic and butyric 230
acid. 231
Table 4 also shows the FEs of the four SCFAs, along with the combined FE. The 3 h 232
and continuous feeding strategies resulted in the highest combined FE, while the lowest 233
occurred with the 24 h feeding method. This is consistent with the lower acid utilization rates 234
observed with the 24 h feeding strategy. 235
In comparing the individual acids, lactic acid was consumed completely, with greater 236
than 95 % utilization of propionic and butyric acids. FEs were higher for all the acids in the 237
bioreactor trials compared to the prior shake flask trials (Table 5). 238
Cell dry weights and PHA production parameters for the different ARF feeding 239
strategies are provided in Table 4. The 3 h feeding strategy resulted in the highest cell dry 240
weight, PHA concentration, and PHA content, but the values were not significantly different 241
from the other feeding strategies. Only the PHA productivity for the 3h feeding strategy was 242
significantly higher. Cell dry weight and PHA production were much higher than that 243
obtained during the shake flask trials. The highest PHA concentration in the cells in the shake 244
flask trials was 4.6 g L-1 (Table 5), whereas in the fermenter trials the PHA concentration of 245
the cells was about 1.82 times higher [4]. 246
247
248
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DISCUSSION 249
Typically, PHA production occurs during stationary phase. Hence cells are first grown 250
through exponential phase in a balanced medium to maximize growth rate. This medium is 251
formulated to run out of a key nutrient when the maximum cell population is achieved, then 252
additional carbon is added by fed-batch or continuous mode to maximize PHA production 253
[19]. 254
In this study nitrogen deficiency was used to trigger PHA production in the presence of 255
excess carbon. Nitrogen supplemented CCS medium resulted in better growth of R. eutropha 256
in the bioreactors as compared to the shake flasks due to the improved aeration and agitation 257
provided in the bioreactor, along with more consistent pH control. The organism continued to 258
grow after the SCFAs were added at 48h. This growth was supported by the residual ammonia 259
and phosphorus present in the medium. The organism reached its stationary phase by 96 h 260
when most of the ammonia and phosphorous present in the medium were consumed. Ideally, 261
one or both of these nutrients becomes limiting at the end of exponential phase, to trigger the 262
shift from reproductive metabolism to PHA synthesis [19]. Because nitrogen and phosphate 263
were not depleted until 72 h, this could have contributed to the continued increase in cell 264
numbers observed after 48 h. It is likely that at least some of the SCFAs fed at 48 h were 265
utilized for growth, until the point at which nitrogen became limiting. Researchers have found 266
that the complete lack of nitrogen may suppress enzyme activity in PHA synthesis [28]. Thus 267
a small amount of ammonia in the media might be necessary to trigger PHA synthesis. 268
The utilization rate of lactic acid in the initial 48h was lower than that of acetic acid 269
probably due to higher concentration of lactic acid (1.6 g L-1) in the bioreactor media 270
13
compared to the shake flask media (1g L-1). This variation was due to the different batches of 271
CCS obtained from the ethanol plant. 272
In this study, fed batch (24h feeding) and continuous feeding strategies were chosen to 273
determine their effect on PHA production. The lowest acid utilization rates and FEs were 274
observed for the 24 h feeding strategy due to the periodic spikes in SCFA concentrations that 275
might have disrupted the acid utilization and decreased cell activity. At high SCFA 276
concentration the pH of the medium can reach below the pKas for SCFAs (lactic 3.86, acetic 277
4.76, butyric 4.83, propionic 4.87). At low pHs the undissociated form predominates, and the 278
SCFAs readily cross the cell membrane. Once inside they rapidly dissociate and acidify the 279
cytoplasm [25]. As a result, the proton gradient cannot be maintained as desired, and energy 280
generation and transport system dependent on proton gradient are disrupted [14]. This can 281
also cause an increase in osmotic pressure due to the accumulation of anions [23]. At pH 282
levels closer to the optimum for R eutropha (~7.0), SCFAs would be in the dissociated form 283
in the medium. While the anions wouldn’t be transported as readily, once inside the cell they 284
would not cause the adverse effects of the undissociated form. Therefore, SCFAs can only be 285
effective carbon sources when pH and SCFA concentrations are carefully regulated. Acid 286
utilization rates might have also been reduced by depletion of certain acids at the end of each 287
24 h phase. 288
For the continuous strategy, small volumes of the SCFAs were fed continuously. 289
Though FEs of the SCFAs were higher than that of the 24h feeding strategy, there were s light 290
accumulations in SCFAs towards the end of the fermentation. 291
It was thus necessary to develop an optimal feeding strategy which could potentially 292
increase acid utilizations and FEs. The 3h feeding strategy was thus chosen. Since the SCFAs 293
14
were added in smaller doses at shorter intervals, pH of the medium was more efficiently 294
regulated, which resulted in better utilization and 100% FE of the organic acids. 295
In comparison to the prior aerated shake flasks trials (Table 5), which were fed with 296
individual SCFAs, the addition of mixed acids to the bioreactor resulted in more rapid uptake 297
of propionic acid and slower utilization of butyric acid. Propionate utilization rate rose from 298
0.046 g L-1 h-1 in aerated shake flasks [4] to approximately 0.08 g L-1 h-1 when added as a part 299
of the ARF mixture in the bioreactor trials. This was likely due to the higher cell populations 300
achieved in the bioreactor trials coupled with the fact that proprionate utilization by R. 301
eutropha is energetically favorable [36]. Moreover, addition of propionic acid in small doses 302
might have controlled the change in pH and thus resulted in better utilization. The decline in 303
the utilization rate of butyric acid, from 0.041 g L-1 h-1 when fed individually in aerated shake 304
flasks to 0.011 g L-1 h-1 in bioreactor trials, may have been due to additional ATP needed to 305
transport this acid [26, 27]. Thus utilization of other acids was preferred over butyric acid 306
when fed as a mixture for growth. 307
The high FEs for lactic and propionic acids were consistent with the metabolic 308
preference of R. eutropha, especially considering that the ARF contained 15 and 20 g L-1 of 309
these acids, respectively. The high FE of butyric acid may be due to the fact that ARF 310
contained only 2 g L-1 of this acid. While conducting the shake flask trials, we had previously 311
noted that R. eutropha did not prefer acetic acid, therefore its lower FE was not surprising. 312
Thus it can be concluded that in the 24h strategy the sudden rise in the SCFA 313
concentrations must have lowered the pH by accumulation of acid. This might have caused 314
decrease in acid consumption, FE and PHA productivity. Though the dosages of acids were 315
small for the continuous feeding, continuous addition might have lowered the efficiency of 316
15
the process. The smaller dosage and the fed batch mode of the 3h feeding strategy might have 317
resulted in better control of pH and of catabolite repression. So the optimized growth 318
conditions (as discussed before) for the organism and 3h feeding strategy of ARF addition 319
was considered the best to obtain optimum PHA production by the organism. 320
Yu et al. suggested [36] in their study that an average yield of PHA was 0.39 g g-1 of 321
carbon sources (acetic, butyric and propionic acids). In the current study the highest yield of 322
about 0.2 g g-1 was observed when the 3h feeding strategy was used. Investigators have 323
reported that inexpensive carbon sources lead to low PHA productivity due to inefficient 324
utilization of nutrients by organisms [16, 32, and 33]. A majority of carbon sources was 325
probably utilized to generate energy for cell maintenance. 326
Wild type strain of R. eutropha used in this study was not able to use glucose and did 327
not completely utilize the high amount of glycerol present in the CCS medium. Recombinant 328
microorganisms can be used to increase growth rate and nutrient utilization. PHA biosynthesis 329
genes can be inserted in organisms that have wider range of utilizable substrates to increase 330
PHA productivity and PHA content. For example, the recombinant strains of R. eutropha 331
(H16) containing glucose-utilizing genes of Escherichia coli (E.coli), and E. coli harboring 332
the R. eutropha genes had higher PHA productivity and concentration as compared to the wild 333
type strain [15,16]. 334
CONCLUSION 335
Since the carbon source is a major contributor to PHA cost, inexpensive sources of 336
carbon are important. From an economical point of view, the use of purified media to increase 337
PHA yield will significantly increase the production cost. This study showed that R. eutropha 338
is capable of growing in CCS medium, a low value byproduct of ethanol industry. We also 339
16
concluded that mixture of SCFAs in the same compositional ratios of ARF was not inhibitory 340
when added at stationary phase of growth. SCFAs can be diverted toward PHA production by 341
R. eutropha. As the use of purified SCFAs is cost prohibitive, developing a mixed culture 342
system to produce a mixture of SCFAs from another ethanol industry byproduct is highly 343
cost-effective. Thus utilization of inexpensive carbon sources may lead to economically 344
viable PHA production in future when superior genetics and fermentation strategies are 345
applied together. 346
Acknowledgement 347
This project was funded by South Dakota Corn Utilization Council and the 348
Agricultural Experiment Station. 349
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444
445
446
447
448
449
450
451
21
TABLES 452
Table 1. CCS composition from a dry mill ethanol plant 453
Components CCS
Dry matter % 34.9
Crude Protein % 13.7
Crude Fat (Ether extract) % 16.2
Ash % 9.5
Crude Fiber % 0.8
Copper ppm 8.3
Sodium ppm 5,620
Calcium ppm 487
Magnesium ppm 6,850
Zinc ppm 49
Phosphorus ppm 15,400
Potassium ppm 22,900
Composition is on dry matter basis 454 455
Table 2 Average growth and nutrient utilization rates of R. eutropha in CCS medium 456
through 48h in biorector. 457
458
Maximum Cell
Population (cfu ml-1)
Maximum Growth
Rate (h-1)
Ammonia Utilization
Rate
(mg L-1 h-1)
Phosphate
Utilization Rate
(mg L-1h-1)
2.3 x 1010 0.20 2.3 0.023 459
SCFA Utilization Rate (gL-1h-1)
Acetic Butyric Lactic Propionic Succinic
0.051 0.026
0.046 0.023 0.027
SCFA FE (%)
Acetic Butyric Lactic Propionic Succinic
100 100 81.25 65.5 71.2
460
22
Table 3 Average growth and nutrient utilization rates of R. eutropha in CCS medium 461
through 48h in shake flasks. 462
463
Maximum Cell
Population (cfu ml-1)
Maximum
Growth Rate (h-1)
Ammonia
Utilization Rate
(mg L-1 h-1)
Phosphate Utilization
Rate
(g L-1 h-1)
2.6×109 0.13 1.7 0.022 464
SCFA Utilization Rate (g L-1 h-1)
Acetic Butyric Lactic Propionic Succinic
0.033 0.026 0.054 0.010 0.021 SCFA Fermentation Efficiency (%)
Acetic Butyric Lactic Propionic Succinic
77.6 76.2 94.2 35.6 62.7
465
466
Table 4 Comparison of all the key parameters under different ARF feeding strategies. 467
468
Key parameters
Feeding strategies
24 h Addition 3 h Addition Continuous
Addition
Cell Population at 48 h
(CFU-1ml) 4.28 x 1010a 1.70 x 1010b 1.01 x1010b
Maximum Cell
Population (CFU-1ml)
5.03 x 1010a 5.68 x 1010a 5.47 x 1010a
Ammonia Utilization
Rate (g-1L-1h)
1.2a 1.0 a 1.1a
Phosphate Utilization
Rate (g-1L-1h) 0.010a 0.013a 0.011a
Acid Utilization (g-1L-1h) Acetic 0.029a 0.052b 0.047b
Butyric 0.009a 0.010a 0.014a Lactic 0.074a 0.080a 0.078a Propionic 0.077a 0.083a 0.080a
Combined 0.20a 0.25b 0.24b Fermentation Efficiency
(%)
Acetic 58.8a 100b 98.7b Butyric 94.7a 100b 100b
Lactic 100a 100a 100a
23
Propionic 95.7a 100b 95a
Combined 82.7a 100b 97.8b PHA Production
Cell Dry Weight (g-1L) 17.6a 21.13a 17.3a PHA Concentration
(g-1L) 6.42a 8.37a 6.67a
PHA Productivity
(g-1L-1h) 0.0537a 0.0697b 0.056a
PHA Content (%) 36.23a 39.52a 37.78a a,b,c Means within column not sharing common superscript differ significantly (p<0.05) 469
Table 5 Comparison of combined short fatty acid feeding of Ralstonia eutropha in shake 470
flasks 471
Parameters
Volatile Fatty Acid
Acetic
(5g L-1)
Butyric
(5g L-1)
Lactic
(8g L-1)
Propionic
(5g L-1)
Maximum Cell Population (cfu ml-1) 5.70 ×109a 6.17 ×109 ab 5.32 ×109a 6.67 ×109b
Fermentation Efficiency (%) 70.6a 95.6b 70.7a 68.6a
Acid Utilization Rate (g L-1 h-1) 0.048a 0.041a 0.080b 0.046a
PHA Concentration (g L-1) 2.9ab 4.6a 2.4b 4.3a
Cell Dry Weight (g L-1) 9.9ab 14.5b 6.0a 14.7b
PHA Productivity (g L-1 h-1)) 0.024ab 0.037a 0.020b 0.036a
PHA Content (%) 29.2a 31.9a 30.7a 29.3a a,b Means within column not sharing common superscript differ significantly (p<0.05). 472
FIGURES 473
474
24
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 5 10 15 20 25 30 35 40 45 50
Time (h)
Co
nce
ntr
ati
on
(g
L-1
)
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
Cel
l P
op
ula
tio
n (
cfu
ml-1
)
475
Figure 1 Average growth rate and organic acid utilization during the initial 48 h 476
incubation in the CCS medium. The values for average cell count (■), acetic acid 477
utilization (Δ), butyric acid utilization (◊), lactic acid utilization (□) propionic acid 478
utilization (▲) and succinic acid utilization (×) are indicated. Values are the means of 479
two replications with standard deviation showed by error bars. 480
481
25
0
1
2
3
4
5
6
7
8
9
0 20 40 60 80 100 120 140
Time (h)
Co
nce
ntr
ati
on
(g
L-1
)
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
Cel
l P
op
ula
tio
n (
cfu
ml-1
)
482
483
484
Figure 2 Acid utilization and growth of Ralstonia eutropha with 24 h interval additions 485
of ARF. The values for cell count (■), acetic acid utilization (Δ), butyric acid utilization 486
(◊), lactic acid utilization (□), propionic acid utilization (▲), succinic acid utilization (×) 487
and PHA concentration (O) are indicated. Values are the means of two replications with 488
standard deviation showed by error bars. 489
490
491
492
493
26
0
1
2
3
4
5
6
7
8
9
0 20 40 60 80 100 120 140
Time (h)
Co
nce
ntr
ati
on
(g
L-1
)
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
Cel
l P
op
ula
tio
n (
cfu
ml-1
)
494
495
Figure 3 Acid utilization and growth of Ralstonia eutropha with 3 h interval additions of 496
ARF. The values for cell count (■), acetic acid utilization (Δ), butyric acid utilization (◊), 497
lactic acid utilization (□), propionic acid utilization (▲), succinic acid utilization (×) and 498
PHA concentration (O) are indicated. Values are the means of two replications with 499
standard deviation showed by error bars. 500
501
502
503
504
505
27
0
1
2
3
4
5
6
7
8
9
0 20 40 60 80 100 120 140
Time (h)
Co
nce
ntr
ati
on
(g
L-1
)
1.00E+06
1.00E+07
1.00E+08
1.00E+09
1.00E+10
1.00E+11
Cel
l P
op
ula
tio
n (
cfu
ml-1
)
506
Figure 4 Acid utilization and growth of Ralstonia eutropha with continuous ARF 507
addition. The values for cell count (■), acetic acid utilization (Δ), butyric acid utilization 508
(◊), lactic acid utilization (□), propionic acid utilization (▲), succinic acid utilization (×) 509
and PHA concentration (O) are indicated. Values are the means of two replications with 510
standard deviation showed by error bars. 511
512
513
514
515
516
517
28
518
519
520
521
522
523
524
525
526
527
528
529
530
531
532
533
534
535
536
537 538